The overall goal of this project is to understand how the molecular motor dynein is regulated to produce the high beat frequency and complex waveforms characteristic of motile cilia and flagella. In mammals, motile cilia / flagella are required for sperm propulsion, removal of debris from the respiratory tract and middle ear, circulation of cerebrospinal fluid, and for determination of the left-right body plan during development. As a consequence, defects in ciliary motility result in impaired fertility, respirator distress, hydrocephalus, otitis media, and/or randomization of the left-right body axis. While significant progress has been made in understanding the force generating properties of the dynein motors, how microtubule sliding is controlled both spatially and temporally remains one of the biggest unanswered questions in the field. Substantial evidence indicates that the central apparatus and radial spokes are major components of a signal transduction network which controls microtubule sliding and ciliary beating. Significant efforts from our lab and others have established the composition of these structures, yet, integrating these components into a broader mechanistic understanding of ciliary motility is lacking. In this proposal, we take a fundamental step towards bridging this gap. In Aims 1 and 2 of the proposal we will capitalize on our discovery that the two radial spokes (RS1 and RS2) of the repeating spoke pairs are heterogeneous in composition. In Aim 1 our identification of the microtubule binding adaptors for RS1 and RS2 provide us with the opportunity to define a mechanism for the targeting and anchoring of specific spoke associated proteins that likely establish the 96 nm axonemal repeat. In Aim 2 we test the hypothesis that each spoke in the pair controls the activity of specific dynein arm subforms. This Aim is supported by our discovery that the adaptor for RS2, the CSC, is required for WT motility and makes contact with the dynein regulatory complex and specific inner dynein arm isoforms. In Aim 3 we focus on radial spoke - central apparatus interactions. This Aim is founded on our discovery of complexes associated with the C1 microtubule of the central apparatus that are essential for wild-type motility. We will combine genetics, functional assays and biophysical /computational approaches to identify key interactions between the central pair projections and radial spokes and to test hypotheses about how physical interactions between these structures modulate microtubule sliding and ciliary beating. Experiments in Aim 3 will likely reveal new principles for the frictional forces acting ata nanoscopic scale in biological systems but which have profound consequences on cell function. Our combined studies will bridge major gaps in our understanding of ciliary motility by addressing fundamental question about how spatially and temporally controlled interactions between large, highly conserved, macromolecular assemblies regulate dynein-driven microtubule sliding. These studies will also have an impact on the more general field of microtubule-associated motors.